Kiefer Patrick, Portais Jean-Charles, Vorholt Julia A
Institute of Microbiology, ETH Zurich, Wolfgang-Pauli-Strasse 10, Hoenggerberg HCI F429, 8093 Zurich, Switzerland.
Anal Biochem. 2008 Nov 15;382(2):94-100. doi: 10.1016/j.ab.2008.07.010. Epub 2008 Jul 18.
In this report, we introduce a liquid chromatography single-mass spectrometry method for metabolome quantification, using the LTQ Orbitrap high-resolution mass spectrometer. Analytes were separated with hydrophilic interaction liquid chromatography. At a working resolution of 30,000 (at m/z 400), the limit of detection varied from 50 fmol to 5 pmol for 25 metabolites tested. In terms of metabolite concentration, the linearity was about 2 to 3 orders of magnitude for most compounds (R(2)>0.99). To determine the accuracy of the system in complex sample matrices, the isotope dilution method was evaluated from mixtures of pure compounds and uniformly 13C-labeled cell extracts. With the application of this method, quantification was possible within single runs even when the pool sizes of individual metabolites varied from 0.13 to 55.6 microM. As a case study, intracellular concentrations of central metabolites were determined for Methylobacterium extorquens AM1 during growth on two different carbon sources, methanol and succinate. Reproducible results from technical and biological repetitions were obtained that revealed significant variations of intracellular metabolite pool sizes, depending on the carbon source. The LTQ Obitrap offers new perspectives and strategies for metabolome quantification.
在本报告中，我们介绍了一种使用LTQ Orbitrap高分辨率质谱仪进行代谢组定量的液相色谱-单重质谱法。分析物通过亲水相互作用液相色谱进行分离。在30,000（m/z 400时）的工作分辨率下，对于所测试的25种代谢物，检测限在50飞摩尔至5皮摩尔之间变化。就代谢物浓度而言，大多数化合物的线性范围约为2至3个数量级（R(2)>0.99）。为了确定该系统在复杂样品基质中的准确性，从纯化合物和均匀13C标记的细胞提取物的混合物中评估了同位素稀释法。应用该方法，即使单个代谢物的库大小在0.13至55.6微摩尔之间变化，也可以在单次运行中进行定量。作为一个案例研究，测定了甲基营养型芽孢杆菌AM1在以甲醇和琥珀酸两种不同碳源生长期间细胞内中心代谢物的浓度。获得了技术和生物学重复的可重复结果，这些结果揭示了细胞内代谢物库大小的显著变化，这取决于碳源。LTQ Obitrap为代谢组定量提供了新的视角和策略。
